Targeting senescence as an anti-cancer therapy

Cellular senescence is a stress response elicited by different molecular insults. Senescence results in cell cycle exit and is characterised by multiple phenotypic changes such as the production of a bioactive secretome. Senescent cells accumulate during ageing and are present in cancerous and fibrotic lesions. Drugs that selectively kill senescent cells (senolytics) have shown great promise for the treatment of age-related diseases. Senescence plays paradoxical roles in cancer. Induction of senescence limits cancer progression and contributes to therapy success, but lingering senescent cells fuel progression, recurrence, and metastasis. In this review, we describe the intricate relation between senescence and cancer. Moreover, we enumerate how current anti-cancer therapies induce senescence in tumour cells and how senolytic agents could be deployed to complement anticancer therapies. "One-two punch" therapies aim to first induce senescence in the tumour followed by senolytic treatment to target newly exposed vulnerabilities in senescent tumour cells. "One-two punch" represents an emerging and promising new strategy in cancer treatment. Future challenges of "one -two punch" approaches include how to best monitor senescence in cancer patients to effectively survey their efficacy

Leaf Eh and pH: A Novel Indicator of Plant Stress. Spatial, Temporal and Genotypic Variability in Rice (Oryza sativa L.)

A wealth of knowledge has been published in the last decade on redox regulations in plants. However, these works remained largely at cellular and organelle levels. Simple indicators of oxidative stress at the plant level are still missing. We developed a method for direct measurement of leaf Eh and pH, which revealed spatial, temporal, and genotypic variations in rice. Eh (redox potential) and Eh@pH7 (redox potential corrected to pH 7) of the last fully expanded leaf decreased after sunrise. Leaf Eh was high in the youngest leaf and in the oldest leaves, and minimum for the last fully expanded leaf. Leaf pH decreased from youngest to oldest leaves. The same gradients in Eh-pH were measured for various varieties, hydric conditions, and cropping seasons. Rice varieties differed in Eh, pH, and/or Eh@pH7. Leaf Eh increases and leaf pH decreases with plant age. These patterns and dynamics in leaf Eh-pH are in accordance with the pattern and dynamics of disease infections. Leaf Eh-pH can bring new insight on redox processes at plant level and is proposed as a novel indicator of plant stress/health. It could be used by agronomists, breeders, and pathologists to accelerate the development of crop cultivation methods leading to agroecological crop protection

The role of Galectin-3 in α-synuclein-induced microglial activation

Background: Parkinson ’ s disease (PD) is the most prevalent neurodegenerative motor disorder. The neuropathology is characterized by intraneuronal protein aggregates of α -synuclein and progressive degeneration of dopaminergic neurons within the substantia nigra. Previous studies have shown that extracellular α -synuclein aggregates can activate microglial cells, induce inflammation and contribute to the neurodegenerative process in PD. However, the signaling pathways involved in α -synuclein-mediated microglia activation are poorly understood. Galectin-3 is a member of a carbohydrate-binding protein family involved in cell activation and inflammation. Therefore, we investigated whether galectin-3 is involved in the microglia activation triggered by α -synuclein. Results: We cultured microglial (BV2) cells and induced cell activation by addition of exogenous α -synuclein monomers or aggregates to the cell culture medium. This treatment induced a significant increase in the levels of proinflammatory mediators including the inducible Nitric Oxide Synthase (iNOS), interleukin 1 Beta (IL-1 β ) and Interleukin-12 (IL-12). We then reduced the levels of galectin-3 expression using siRNA or pharmacologically targeting galectin-3 activity using bis-(3-deoxy-3-(3-fluorophenyl-1 H -1,2,3-triazol-1-yl)- β -D-galactopyranosyl)-sulfane. Both approaches led to a significant reduction in the observed inflammatory response induced by α -synuclein. We confirmed these findings using primary microglial cells obtained from wild-type and galectin-3 null mutant mice. Finally, we performed injections of α -synuclein in the olfactory bulb of wild type mice and observed that some of the α -synuclein was taken up by activated microglia that were immunopositive for galectin-3. Conclusions: We show that α -synuclein aggregates induce microglial activation and demonstrate for the first time that galectin-3 plays a significant role in microglia activation induced by α -synuclein. These results suggest that genetic down-regulation or pharmacological inhibition of galectin-3 might constitute a novel therapeutic target in PD and other synucleinopathie

The SECURE project – Stem canker of oilseed rape: : molecular methods and mathematical modelling to deploy durable resistance

N Evans et al, "The SECURE Project - Stem Canker of oilseed rape: Molecular methods and mathematical modeling to deploy durable resistance", in Vol 4 of the Proceedings of the 12th International Rapeseed Congress : Sustainable Development in Cruciferous Oilseed Crops Production, Wuhan, China, March 26 - 30, 2007. The proceedings are available online at: http://gcirc.org/intranet/irc-proceedings/12th-irc-wuhan-china-2007-vol-4.htmlModelling done during the SECURE project has demonstrated the dynamic nature of the interaction between phoma stem canker (Leptosphaeria maculans), the oilseed rape host (Brassica napus) and the environment. Experiments done with near-isogenic lines of L. maculans to investigate pathogen fitness support field data that suggest a positive effect of the avirulence allele AvrLm4 on pathogen fitness, and that the loss of this allele renders isolates less competitive under field conditions on cultivars without the resistance gene Rlm4. The highlight of molecular work was the cloning of AvrLm1 and AvrLm6. L. maculans is now one of the few fungal species for which two avirulence loci have been cloned. Subsequent research focused on understanding the function of AvrLm1 and AvrLm6 and on the analysis of sequences of virulent isolates to understand molecular evolution towards virulence. Isolates of L. maculans transformed with GFP and/or DsRed were used to follow growth of the fungus in B. napus near-isogenic-lines (NIL) with or without MX (Rlm6) resistance under different temperature and wetness conditions. The results greatly enhanced our knowledge of the infection process and the rate and extent of in planta growth on different cultivars. Conclusions from work to model durability of resistance have been tested under field conditions through a series of experiments to compare durability of resistance conferred by the major resistance gene Rlm6 alone in a susceptible background (EurolMX) or in a resistant background (DarmorMX) under recurrent selection over 4 growing seasons. A major priority of the project was knowledge transfer of results and recommendations to target audiences such as plant breeding companies and extension services. CETIOM developed a “diversification scheme” that encourages French growers to make an informed choice about the cultivars that are grown within the rotation based on the resistance genes carried by the individual cultivars. Use of such schemes, in association with survey data on the population structure of L. maculans at both national and European scales will provide opportunities for breeders and the industry to manage available B. napus resistance more effectively.Non peer reviewe

Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis

BACKGROUND: A number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2. CONCLUSIONS/SIGNIFICANCE: The results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases

C9ORF72-derived poly-GA DPRs undergo endocytic uptake in iAstrocytes and spread to motor neurons

Dipeptide repeat (DPR) proteins are aggregation-prone polypeptides encoded by the pathogenic GGGGCC repeat expansion in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic β-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species

Distinct tau prion strains propagate in cells and mice and define different tauopathies

Prion-like propagation of tau aggregation might underlie the stereotyped progression of neurodegenerative tauopathies. True prions stably maintain unique conformations (“strains”) in vivo that link structure to patterns of pathology. We now find that tau meets this criterion. Stably expressed tau repeat domain indefinitely propagates distinct amyloid conformations in a clonal fashion in culture. Reintroduction of tau from these lines into naive cells reestablishes identical clones. We produced two strains in vitro that induce distinct pathologies in vivo as determined by successive inoculations into three generations of transgenic mice. Immunopurified tau from these mice recreates the original strains in culture. We used the cell system to isolate tau strains from 29 patients with 5 different tauopathies, finding that different diseases are associated with different sets of strains. Tau thus demonstrates essential characteristics of a prion. This might explain the phenotypic diversity of tauopathies and could enable more effective diagnosis and therapy

[SWI+], the Prion Formed by the Chromatin Remodeling Factor Swi1, Is Highly Sensitive to Alterations in Hsp70 Chaperone System Activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments

Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms